Metagenomics of Dental Caries: A Polymicrobial Disease
Dental caries , more commonly known as tooth decay or cavities, is the destruction of enamel, dentin or the cementum caused by acid producing bacteria in a dental plaque. The result is a lesion in the crown or the root surface of the tooth. Unlike most infectious diseases where there is a single etiological agent, dental caries is caused by a host of bacteria and, therefore, is termed a polymicrobial disease. Because many of the bacteria that inhabit the human oral cavity cannot be grown in culture, many researchers have tried studying the oral microbiome through metagenomic studies in order to identify the diverse species of bacteria that inhabit the diseased and healthy mouth. History Caries were very uncommon in fossil hominids into the Paleolithic (characterized by the development of stone tools and formation of bands; 2.6 million years ago to 10,000 BC) and Mesolithic (characteristic for agriculture and more sedentary populations; 10,000 BC to 5,000 BC). It is estimated that only 1% of ancient hominids had dental caries. In European populations, the incidence of dental caries was sporatic throughout and up until 10,000 AD, around the time when sugar was introduced (3). Now, about 15.6% of children 6-19 years old and 23.7% of adults 20-64 years old have untreated dental caries (4). Metagenomic Studies Introduction It has been demonstrated that Streptococcus mutans and Streptococcus sobrinus are acid producing bacteria that play a major role in dental caries (1). However, there is indications that S. mutans is not the only bacteria necessary for the onset of caries. In preliminary data, about 10% of subjects with rampant dental caries in permanent teeth had no detectable levels of S. mutans ''(2). Aas et al. set out with two goals: 1) to determine all bacteria species associated with health and dental caries, and 2) to describe the changes in microbiota during different stages of the disease. Methods: Reverse-capture checkerboard assay #Subject population and sampling: Subjects with severe primary (n=15) and secondary tooth dental caries (n=36) and age-matched caries free controls were recruited from The Columbus Children's Hospital Dental Clinic. Plaques from the healthy controls were pooled from four sites, including anterior and posterior teeth. From the caries patients, plaques were pooled from surfaces of intact enamel, surfaces of white-spot lesions, cavited dentin lesions and deep dentin lesions. #Isolation of bacteria DNA #Subjects for clonal analysis: Three caries subjects with detectable ''S. mutans, two caries subjects without detectable S. mutans and controls. #Amplification of 16S rRNA by PCR and cloning #'Reverse-capture checkerboard assay': A reverse-capture checkerboard assay is a hybridization assay that is used for the direct detection of bacterial species (5). Species specific capture probes that target 16S rRNA are attached to a nylon membrane. 16S rRNA genes from the plaque and cavity samples are amplified prior by PCR using a digoxin labeled primer. Hybridizations are performed in vertical channels with a Miniblotter and digoxin amplicons. Hybridization reactions are detected by chemifluorescence procedure. Bacteria numbers are determined using a Storm system. A genus specific probe allows one to assess the total number of a specific genus, say streptococci, whereas species wide probes assess total numbers of bacteria. Capture probes in this study were designed for the most prevalent bacterial species. See here for the full list of bacteria that probes were generated for. #Analysis: Bacterial signals were measured by the intensity of spots on the reverse-capture checkerboard assay membrane with ImageQuant TL. Results This study found that there were 197 species, or phylotypes, representing 8 different bacterial phyla that inhabit the diseased and/or healthy mouth. Of these, Aas et al. found 12 previously uncharacterized strains, 99 strains (50%) that had not been previously cultured and 86 known species. In total, 22 novel species were identified. Goal 1: To determine all the bacteria that inhabit dental caries and healthy individuals: see Figure 1. Goal 2: To determine which microbiota inhabit the dental caries depending on the stage of the disease: see Figure 2, Figure 3, Figure 4 and Figure 5. For a summary, see Figure 6, although beware that this table is not very organized. Summary Aas et al. have shown that half of the bacterial species that are associated with dental caries have not yet been cultured in lab, making metagenomic studies prudent to full understanding of dental caries. They also found that in samples with detectable levels of S. mutans, they found that other genera including Atopobium, Lactobacillus and Propionibacterium were present at significantly higher levels. In samples without detectable levels of S. mutans, low-pH non-''S. mutans streptococci, ''Bifidobacterium dentium and Lactobacillus ''were present. ''Actinomyces and non-''S. mutans'' were associated with white spot lesions, whereas known acid producing bacteria were present in later stages of the disease. Importantly, they identified bacteria that are associated only with health and others that are associated only with disease. Futhermore, they succeeded in finding that different bacterial species are present in primary and secondary dentition. There were 15 health associated microbes in primary dentition and in contrast to the 30 health associated microbes in permanent dentition. These findings support the ecological plaque hypothesis, which states that changes in ecological factors require different bacterial qualities and will therefore alter bacterial composition within the oral cavity. References #http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2012.03857.x/full#b2 #http://jcm.asm.org/content/46/4/1407.full #http://www.uic.edu/classes/osci/osci590/11_1Epidemiology.htm #http://www.cdc.gov/nchs/fastats/dental.htm #http://link.springer.com/article/10.1023%2FA%3A1009715710555